Preparation, characterization and transfection efficiency of cationic PEGylated PLA nanoparticles as gene delivery systems.

The cationic polylactic acid (PLA) nanoparticle has emerged as a promising non-viral vector for gene delivery because of its biocompatibility and biodegradability. However, they are not capable of prolonging gene transfer and high transfection efficiency. In order to achieve prolonged delivery of cationic PLA/DNA complexes and higher transfection efficiency, in this study, we used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), PLA and chitosan (CS) to prepare MePEG-PLA-CS NPs and PLA-CS NPs by a diafiltration method and prepared NPs/DNA complexes through the complex coacervation of nanoparticles with the pDNA. The object of our work is to evaluate the characterization and transfection efficiency of MePEG-PLA-CS versus PLA-CS NPs. The MePEG-PLA-CS NPs have a zeta potential of 15.7 mV at pH 7.4 and size under 100 nm, while the zeta potential of PLA-CS NPs was only 4.5 mV at pH 7.4. Electrophoretic analysis suggested that both MePEG-PLA-CS NPs and PLA-CS NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed MePEG-PLA-CS NPs exhibit a low cytotoxicity to normal human liver cells. The potential of PLA-CS NPs and MePEG-PLA-CS NPs as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. The pDNA being carried by MePEG-PLA-CS NPs, PLA-CS NPs and lipofectamine could enter and express in COS7 cells. However, the transfection efficiency of MePEG-PLA-CS/DNA complexes was better than PLA-CS/DNA and lipofectamine/DNA complexes by inversion fluorescence microscope and flow cytometry. It was distinctively to find that the transfection activity of PEGylation of complexes was improved. The nanoparticles were also tested for their ability to transport across the gastrointestinal mucosa in vivo in mice. In vivo experiments showed obviously that MePEG-PLA-CS/DNA complexes mediated higher gene expression in stomach and intestine of BALB/C mice compared to PLA-CS/DNA and lipofectamine/DNA complexes. These results suggested that MePEG-PLA-CS NPs have favorable properties for non-viral gene delivery.

Effect of poly(ethylene glycol)-block-polylactide nanoparticles on hepatic cells of mouse: low cytotoxicity, but efflux of the nanoparticles by ATP-binding cassette transporters.

The objective of this paper is to study the effects of poly(ethylene glycol)-block-polylactide (PLA-PEG) nanoparticles on hepatic cells of mouse. Blank PLA-PEG nanoparticles have been successfully prepared and MTT assay suggested that the nanoparticles with HepG2 cell co-culture model did not cause significant changes in membrane integrity in controlled concentration range (0.001-0.1 mg/ml). Immunohistochemical analysis demonstrated that large dose of PLA-PEG nanoparticles injection (42.04 mg/kg, i.v.) did not induce hepatic cell apoptosis. From biochemical assay experiments, although the levels of SOD decreased and those of MDA, NOS increased after treatment with large dose of PLA-PEG nanoparticles injection (42.04 mg/kg, i.v.), they were all not significant (p>0.05). Then Kunming mice were treated with large dose of PLA-PEG nanoparticles (42.04 mg/kg, i.v.) and after 4 days total RNA was isolated to elucidate patterns of gene expression using a mouse cDNA-microarray (SuperArray). Treatment with nanoparticles resulted in over-expression of a lot of ATP-binding cassette (ABC) transporters, especially two ABC transporters (ABCA8 and ABCC5/MRP5), and down-regulation of GSTP1, in comparison with the control. ABCA8 could extrude low molecular weight polymers after PLA-PEG nanoparticles hydrolysis outside the cells. We also discovered that ABCC5 expressed multidrug resistance protein 5 (MRP5) to pump out conjugate (GS-X) of PLA-PEG nanoparticles with GSH. The results were confirmed by RT-PCR. Results of in vitro accumulation and efflux experiments indicated that about 51-52% (51.5% and 52.0%) intracellular PLA-PEG nanoparticles was expulsed after mouse primary hepatocytes reached a saturation uptake of nanoparticles during the concentration range of 750-1000 microg/ml. The results suggested that ABC transporters (especially ABCA8) pump out the polymers after hydrolysis from mouse hepatic cells and large dose of PLA-PEG nanoparticles make mouse hepatic cells gain drug resistance to PLA-PEG nanoparticles.

A novel PEGylation of chitosan nanoparticles for gene delivery.

CS (chitosan) has emerged as a promising non-viral vector for gene delivery because of its ability to form complexes with pDNA (plasmid DNA) and enhance its transport across cellular membranes through endocytosis. Complexes of CS and pDNA may improve transfection efficiency; however, they are not capable of sustained DNA release and prolonging gene transfer. In order to achieve prolonged delivery of CS-DNA complexes, we prepared CS NP (nanoparticle) and CS-DNA complexes. alpha-Methoxy-omega-succinimidylpoly(ethylene glycol) was then conjugated to the surface of CS-DNA complexes using an active ester scheme; finally, the potential of PEGylation [poly(ethylene glycol)ylation] of CS NP as a non-viral gene-delivery vector to transfer exogenous genes in vitro and in vivo were examined. Electrophoretic analysis suggested that CS NPs could protect the DNA from nuclease degradation. The pDNA carried by CS NPs could enter and be expressed in HepG2 cells. However, the transfection efficiency was very low and the highest dose of DNA transferred was 1.6 microg. The transfection activities of CS-DNA-PEG were preserved and a higher dose (2.4 microg) of pDNA was transferred. This indicated that the transfection efficiency of the PEGylated complexes had been improved. In vivo experiments also showed that CS-DNA-PEG complexes mediated higher gene expression in tissues than did CS-DNA complexes, and that gene expression in tumours induced by CS-DNA-PEG complexes was the highest of all. These results suggested that PEGylation of CS-DNA complexes improves non-viral gene delivery in vitro or in vivo and has the potential to deliver therapeutic genes directly into hepatoma tissues.

Polybutylcyanoacrylate nanoparticles as novel vectors in cancer gene therapy.

To make progress toward an efficient gene vector for cancer gene therapy, a novel nonviral vector of polybutylcyanoacrylate nanoparticles (PBCA NPs) was developed. Cetyltrimethyl ammonium bromide (CTAB) was used to modify the surface of PBCA NPs, and then the plasmid DNA (pDNA) of pAFP-TK was wrapped into PBCA-CTAB NPs. Atomic force microscopy and zeta potential demonstrated that PBCA-CTAB NPs were 80-200 nm in diameter and had +15.6 mV positive surface charges. Assay using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide showed that PBCA-CTAB NPs had less cytotoxicity to 3T3 cells than HepG2 cells. The analysis of PBCA-CTAB-DNA complexes could not only protect DNA from degradation by DNase I, it could also transfer pDNA into targeted cells with high transfection efficiency. Furthermore, when PBCA-CTAB NPs combined with suicide gene pAFP-TK, alpha-fetoprotein-positive cells transfected by it were highly sensitive to ganciclovir treatment, and cell survival declined precipitously. Therefore, this target strategy using a pAFP-TK/GCV suicide gene therapy system in which PBCA-CTAB NPs serve as gene delivery vectors explores a promising area for alpha-fetoprotein-positive hepatocellular carcinoma and associated carcinoma therapy.

Meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles were internalized by SW480 cells by a clathrin-mediated endocytosis pathway to induce high photocytotoxicity.

We studied the uptake of meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles by SW480 cells and carried out a systematic investigation of the cellular internalization mechanism of TCPP nanoparticles, also studied the photocytotoxicity of TCPP nanoparticles. At first, meso-tetra (carboxyphenyl) porphyrin (TCPP) nanoparticles were prepared by the method of mixing solvent techniques. SW480 cellular uptakes of photosensitizers (TCPP nanoparticles, TCPP-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles and free TCPP) were analyzed by the method of fluorospectrophotometry. Endocytosis mechanism investigation was carried out by preincubating SW480 cells at 4 degrees C, and preincubating SW480 cells with sucrose, K+-free buffer solution and filipin. Clathrin HC expression after incubating SW480 cells with these three photosensitizers was analyzed by methods of Western blot and RT-PCR. At last, we analyzed the photo-cytotoxicity after incubating SW480 cells with photosensitizers and receiving irradiation. SW480 cells showed rapid uptake (0.0083fmoles TCPP/cell) of TCPP nanoparticles after 1h incubation. We also demonstrated that the uptake of TCPP nanoparticles by SW480 cells was a clathrin-mediated endocytosis pathway. As a result of rapid internalization of TCPP nanoparticles by SW480 cells, this special photosensitizer showed very high photocytotoxic effect on SW480 cells in vitro. The nano-sized photosensitizer with no matrix cover: TCPP nanoparticles, can produce higher photocytotoxicity than other photosensitizers (TCPP-loaded PLGA nanoparticles and free TCPP). The in vivo tumor growth inhibition experiment indicated that TCPP nanoparticles plus PDT treatment induced the most dramatic tumor-inhibiting efficacy in all TCPP treated groups. The results of this study suggest that TCPP nanoparticles represent a potential and powerful photodynamic therapy agent.

Arg-Gly-Asp (RGD) peptide conjugated poly(lactic acid)-poly(ethylene oxide) micelle for targeted drug delivery.

In this study, a new poly(lactic acid)-poly (ethylene oxide)-Arg-Gly-Asp (PLA-PEO-RGD) derivative was synthesized, and paclitaxel-loaded PLA-PEO-RGD micelles were prepared by this derivative. The solubility assay showed that micelles mixed with Pluronic F-68 as surfactant could increase the solubility of this hydrophobic paclitaxel in aqueous solution. The cell-binding assay showed that PLA-PEO-RGD micelle (IC(50) = 11.13 +/- 1.38 nmol/L) had about 3.6-fold higher integrin avidity than PLA-PEO-RGD conjugates (IC(50) = 40.33 +/- 3.12 nmol/L). The avidity of micelle was also higher than RGD4C peptide (IC(50) = 24.44 +/- 1.21 nmol/L). The in vitro drug release profile of drug-loaded PLA-PEO-RGD micelles exhibited initial burst release to 37% +/- 2% (w/w) during the first 12 h, and then the release rate became steady in a controlled release manner. Furthermore, treatment of the MDA-MB-435 breast cancer cell line with paclitaxel-loaded PLA-PEO-RGD micelles yielded cytotoxicities, with EC(50) values of approximately 30 mumol/L. The paclitaxel-loaded PLA-PEO-RGD micelles treated group showed the most dramatic tumor reduction in MDA-MB-435 tumor-bearing nude mice, and the final mean tumor load was 31 +/- 16 mm(3) (mean +/- SD; n = 8). (125)I-labeled micelles administration resulted in significant (p < 0.001) higher tumor uptake (2.68% +/- 0.14%, ID/g) of PLA-PEO-RGD micelles compared to PLA-PEO micelles (0.84% +/- 0.09%, ID/g) after 2.5 h postinjection. Biodistribution study showed the best blood clearance of PLA-PEO-RGD micelles after 4.5 h postinjection. The results of this study suggest that paclitaxel-loaded PLA-PEO-RGD micelles based on the specific recognition of alpha(V)beta(3) integrin represent a potential and powerful target delivery technology.